Group A rotavirus (RV) is the main cause of severe diarrhoea in children and in the young of many animal species of economic interest (bovines, porcines, equines, South American camelids, etc.). As a public health problem, RV is the third most common cause of death associated with severe diarrhoea in developing countries (2 million deaths per year). On the other hand, RV-induced diarrhoea in animals intended for consumption, for example young calves, causes high costs related to prevention or treatment.
Group A RV are particles composed of a triple protein capsid. The outer capsid surface is composed of proteins VP4 and VP7, both of which are highly variable antigens; thus far, at least 27 variants of VP4 (P-types) and 16 variants of VP7 (G-types) have been described. Each G-P type combination induces neutralising antibodies that have low cross-reactivity with other G-P types; for this reason, it is necessary to include the different strains that circulate in the target species in the vaccines.
The intermediate capsid is composed of trimeric protein VP6, which represents 51% of the virion mass. Depending on the presence or absence of two different epitopes in protein VP6 (recognised by monoclonal antibodies mAb 255/60 and 631/9), Group A RV strains are additionally classified as subgroups (Sb) I, II, I+II and noI noII. Human RV are usually Sb II, whereas animal RV are primarily Sb I. Protein VP6 is highly immunogenic; naturally infected humans and animals develop a strong humoral response against VP6 epitopes. Regardless of the above-mentioned subgroups, VP6 is a highly conserved protein within all the Group A RV (>90% amino acid homology), and the shared common antigens may be detected by broadly reactive polyclonal antisera or monoclonal antibodies. Therefore, VP6 is the target antigen in most immunodiagnostic tests designed to detect Group A RV. The antibodies directed against this protein do not have neutralising activity in vitro. However, IgA monoclonal antibodies manage to block viral replication intracellularly in mice.
Currently, the prevention of RV-induced diarrhoea in animals is based on passive immunisation, whereas active immunisation is used in human beings. In animals, parenteral inactivated virus vaccines are applied in pregnant females, in order to protect the neonates through the transfer of maternal antibodies via the colostrum and the milk. This strategy is highly effective in preventing the symptoms of severe diarrhoea and in reducing the morbility and mortality in the affected stocks, but it is not capable of preventing RV infection because it does not significantly reduce the amount of virus excreted by the infected animals (Parreno, V. C. et al., Vet Immunol Immunopathol 100:7-24, 2004). Only the continued presence of high titres of passive anti-RV antibodies in the intestinal lumen (naturally produced or artificially added to the milk) completely protects against diarrhoea and significantly reduces viral excretion (Fernandez, F. M. et al., Vaccine 16:507-16, 1998; Saif, L. J. et al., Infect Immun 41:1118-31, 1983, and Saif, L. J. et al., Adv Exp Med Biol 216B:1815-23, 987).
In children, two live virus vaccines attenuated by genetic reassociation have been approved. Both products have proven good efficacy against severe RV-induced diarrhoea. However, given the history of intussusception associated with a vaccine previously used in humans (Murphy, T. V. et al., J Infect Dis 187:1309-13, 2003) and the recent discovery of RV viraemia in naturally infected children (Ray, P. et al., J Infect Dis 194:588-93, 2006, and Blutt, S. E. et al., Lancet 362:1445-9, 2003), the innocuousness of said vaccines has been called into question, specially in premature, immunosuppressed and malnourished children. Therefore, alternative, complementary strategies are needed for the prevention and treatment of RV-induced diarrhoea.
Passive immunity strategies, such as breastfeeding, the administration of anti-RV antibodies purified from bovine colostrum or eggs (human and bovine anti-RV IgG and chicken egg yolk IgY), were shown to reduce diarrhoea disease in both humans and animals. But the possibility of producing large quantities of antibodies in a cost-efficient manner, and with reproducible properties, is low. Therefore, it is necessary to generate antibodies designed for passive anti-RV immunisation in animals and human beings, particularly antibodies that may be produced at industrial scale, that do not cause immunological reactions, that are sufficiently small to efficiently access the epitopes of conserved internal proteins, and that are capable of recognising and inhibiting the replication of strains from different genotypes (polyreactive).
The VHH domain of the camelid antibody heavy chain is, with a weight of 15 kDa, the smallest known natural domain with complete antigen-binding capacity, is ideal to generate encoding DNA libraries for single-chain fragments with a natural antigen recognition capacity. Moreover, strategies to immunise llamas may be used to enrich the VHH library in those directed against an antigen of interest. Due to its particular properties, VHH domains derived from llama antibody heavy chains are very versatile tools for the development of diagnostic reagents and products designed to prevent or treat RV-induced diarrhoea. For example, a VHH directed against a G3 G-type RV strain, produced in yeasts, has recently been reported to show neutralising activity in vitro, and the purified VHH was capable of reducing the occurrence and the duration of RV-induced diarrhoea in lactating mice (Pant, N. et al., J Infect Dis 194:1580-8, 2006, and van der Vaart J. M. et al., Vaccine, May 8, 24(19):4130-7, 2006). However, these authors have not been able to identify against which viral protein the VHH obtained are directed and they assume that they would be directed against conformational epitopes of external proteins.
Patent document WO 2006/056306 discloses the production and use of VHH domains or fragments thereof as a therapy for infections produced by entero-pathogenic microorganisms, for example RV. It shows the production of said VHH or the use thereof in a specific-site release system. For example, it discloses the release of the specific VHH in the gastrointestinal system by encapsulation in alginate. Moreover, as a release method it proposes the use of transgenic probiotic microorganisms which release the specific VHH antibodies and wherein said microorganisms may colonise the human intestine. It proposes different strategies to prepare drugs or foods using VHH antibodies that are encapsulated or expressed by probiotic microorganisms. The VHH produced do not bind to VP6, would not be neutralising, and are also not used by themselves, but within a controlled-release system.
Patent document US 20050054001, by Muyldermans Serge, discloses heavy-chain antibodies, functional domains of heavy-chain antibodies, functional VH domains or fragments thereof which comprise certain modified or mutated amino acid sequences. It does not disclose sequences that correspond to VHH domains which bind to RV VP6.
Patent document WO 00/65057 discloses monovalent proteins that comprise a single variable domain, which bind to viral antigens, particularly bacteriophage P2 of Lactococcus. It only discloses VHH sequences that inhibit said bacteriophage.
Patent document US 2007/0009512, by Hamers et al., and previous documents by the same inventors, disclose heavy-chain fragments of immunoglobulins and the use thereof for veterinary treatments, for example passive immunotherapy or serotherapy. The VHH described only recognise the tetanus toxin. The method used to obtain the VHH is from immunised camelids' mRNA.